Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Intramolecularly hydrogen-bonded organic compounds often exhibit fluorescence emission at considerably longer wavelengths than typical fluorescence as a result of excited-state intramolecular proton transfer (ESIPT). The structure-property relationship of such ESIPT molecules, however, remains obscure. The present article reports the excited-state dynamics of a new family of ESIPT molecules, 2-(2'-hydroxynaphthyl)benzazoles 1-3, based on steady-state and time-resolved spectroscopy measurements. In comparison with the parent compound HBO, all three compounds 1-3 exhibited absorption bands at longer wavelengths and emitted fluorescence from the excited keto-tautomer K* at shorter wavelengths, indicating that the introduction of a naphthalene ring increases the energy gap between the ground and excited states for the keto-tautomer despite the expansion of the aromatic ring. Time-resolved fluorescence spectra revealed dual emission for compounds 1 and 3, consisting of two distinct fluorescence bands originating from K* and the excited rotamer E'*, whereas 2 exhibited fluorescence only from the K* state. In the transient absorption spectra, both the T-T absorption band and the ground state absorption band of the Z-keto tautomer were observed for 1, whereas only the T-T absorption band was observed for 2 and only the Z-keto tautomer band was observed for 3.

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Second harmonic generation (SHG) imaging can be used to visualize unique biological phenomena, but currently available dyes limit its application owing to the strong fluorescent signals that they generate together with SHG. Here we report the first non-fluorescent and membrane potential-sensitive SHG-active organic dye Ap3. Ap3 is photostable and generates SH signals at the plasma membrane with virtually no fluorescent signals, in sharp contrast to the previously used fluorescent dye FM4-64. When tested in neurons, Ap3-SHG shows linear membrane potential sensitivity and fast responses to action potentials, and also shows significantly reduced photodamage compared with FM4-64. The SHG-specific nature of Ap3 allows simultaneous and completely independent imaging of SHG signals and fluorescent signals from various reporter molecules, including markers of cellular organelles and intracellular calcium. Therefore, this SHG-specific dye enables true multimodal two-photon imaging in biological samples.

GABA(γ-amino-butylic acid)-mediated inhibition in the dendrites of CA1 pyramidal neurons was characterized by two-photon uncaging of a caged-GABA compound, BCMACM-GABA, and one-photon uncaging of RuBi-GABA in rat hippocampal slice preparations. Although we found that GABAA-mediated currents were diffusely distributed along the dendrites, currents elicited at the branch points of the apical dendritic trunk were approximately two times larger than those elsewhere in the dendrite. We examined the inhibitory action of the GABA-induced currents on Ca2+ transients evoked with a single back-propagating action potential (bAP) in oblique dendrites. We found that GABA uncaging selectively inhibited the Ca2+ transients in the region adjacent (<20 µm) to the uncaging site, and that GABA uncaging was effective only within a short period after bAP (<20 ms). The strength of inhibition was linearly related to the amplitudes of the GABA currents, suggesting that the currents inhibited a sustained, subthreshold after-depolarization without preventing propagation of bAP. GABA uncaging at the dendritic branch points inhibited Ca2+ transients farther into dendritic branches (>20 µm). Our data indicate that GABA inhibition results in spatially confined inhibition of Ca2+ transients shortly after bAP, and suggest that this effect is particularly potent at the dendritic branch points where GABA receptors cluster.

Heme binds selectively to the 3'-terminal G-quartet (G6 G-quartet) of an all parallel-stranded tetrameric G-quadruplex DNA, [d(TTAGGG)], to form a heme-DNA complex. Complexes between [d(TTAGGG)] and a series of chemically modified hemes possessing a heme Fe atom with a variety of electron densities were characterized in terms of their peroxidase activities to evaluate the effect of a change in the electron density of the heme Fe atom (ρ) on their activities. The peroxidase activity of a complex decreased with a decreasing ρ, supporting the idea that the activity of the complex is elicited through a reaction mechanism similar to that of a peroxidase. In the ferrous heme-DNA complex, carbon monoxide (CO) can bind to the heme Fe atom on the side of the heme opposite the G6 G-quartet, and a water molecule (HO) is coordinated to the Fe atom as another axial ligand, trans to the CO. The stretching frequencies of Fe-bound CO (ν) and the Fe-C bond (ν) of CO adducts of the heme-DNA complexes were determined to investigate the structural and electronic natures of the axial ligands coordinated to the heme Fe atom. Comparison of the ν and ν values of the heme-DNA complexes with those of myoglobin (Mb) revealed that the donor strength of the axial ligation trans to the CO in a complex is considerably weaker than that of the proximal histidine in Mb, as expected from the coordination of HO trans to the CO in the complex.

Key points• The signal for skeletal muscle contraction is a rapid increase in cytosolic Ca 2+ concentration, which requires the coordinated opening of ryanodine receptor (RyR) channels in the sarcoplasmic reticulum.• Channel opening is controlled by voltage-sensing dihydropyridine receptors (DHPRs) of plasma membrane and T tubules. Whether or not their signal is amplified by Ca 2+ -induced Ca 2+ release (CICR) is controversial.• We used two-photon lysis of an advanced Ca 2+ cage to produce local Ca 2+ concentration transients that were large, fast, reproducible and quantifiable, while monitoring the cellular response with a dual confocal laser scanner.• Single frog muscle cells in physiological solutions responded to transients greater than 0.28 μM with propagated CICR waves.• Mouse cells did not respond to stimuli up to 8 μM, unless channel opening drugs were present.• We conclude that CICR contributes to physiological Ca 2+ release in frog but not mouse muscle.• Mice and presumably other mammals do have a capability for CICR that is normally inhibited.It could be manifested under special circumstances, including diseases.Abstract The contribution of Ca 2+ -induced Ca 2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca 2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca 2+ ] rapidly and reversibly, up to 8 μM, levels similar to those reached during physiological activity. They improve over previous paradigms in rate of rise, locality and reproducibility. Use of NDBF-EGTA allowed for the separate modification of resting [Ca 2+ ], trigger [Ca 2+ ] and resting [Mg 2+ ]. In frog muscle, SLICs elicited propagated responses that had the characteristics of CICR. The threshold [Ca 2+ ] for triggering a response was 0.5 μM or less. As this value is much lower than concentrations prevailing near channels during normal activity, the result supports participation of CICR in the physiological control of contraction in amphibian muscle. As SLICs were applied outside cells, the primary stimulus was Ca 2+ , rather than the radiation or subproducts of photorelease. Therefore the responses qualify as 'classic' CICR. By contrast, mouse muscle fibres did not respond unless channel-opening drugs were present at substantial concentrations, an observation contrary to the physiological involvement of CICR in mammalian excitation-contraction coupling. In mouse muscle, the propagating wave had a substantially lower release flux, which together with a much higher threshold justified the absence of response when drugs were not present. The differences in flux and threshold may be ascribed to the absence of ryanodine receptor 3 (RyR3) isoforms in adult mammalian muscle.

The compound 2-[(1E)-2-(1H-pyrrol-2-yl)ethenyl]-quinoxaline (PQX) is a promising fluorescent chromophore for the estimation of protein binding site polarity, due to its full-color solvatochromic fluorescence. A linear relationship was obtained between the peak emission wavenumber and E(T)(N) (normalized solvent polarity). The BSA binding site polarity was estimated from the solvatochromic plot.